The shikimate pathway is remarkably conserved across prokaryotes and eukaryotes to the extent that heterologous transformation of genes associated with the pathway has been successful across highly divergent organisms including bacteria, fungi and plants. Being the only source of aromatic amino acid-precursors for the phenylpropanoid, tryptophan, tyrosine and flavonoid pathways, as well as shuttling between 30-50% of all fixed carbon, the shikimate pathway is a crucial source of structural, defense, light harvesting and hormone signaling molecules essential for plant survival. Additionally, since humans and animals cannot synthesize aromatic amino acids, they are dependent on this pathway as the only dietary source of these essential amino acids. As such, the shikimate pathway has been extensively studied across highly divergent taxa for economic, nutritional and medicinal reasons. In plants, one of the most characterized steps in the shikimate pathway is the sixth reaction that is catalyzed by the enzyme 5-enolpyruylshikimate 3-phosphate synthase (EPSP) synthase. This enzyme catalyzes the conversion of shikimate 3-phosphate to 5-enolpyruvylshikimate 3-phosphate and is the target of the herbicide glyphosate. Isoforms of this enzyme derived from some microbes are naturally resistant to glyphosate and have been used extensively in heterologous transformation to create herbicide resistant plants. No other roles have been assigned for EPSPs outside of catalysis in the shikimate pathway.
Production of renewable fuel from lignocellulosic plant biomass is based on extraction of sugars from plant cell wall material. This extraction process is hampered by the presence of lignin in the cell wall. Lignins contribute to plant “recalcitrance”, a term referring to the inherent resistance of plant material to release polysaccharides and other desirable biomaterials from an interwoven matrix of desirable and undesirable materials (Lynd L R. et al., Science 251:1318-1323 (1991)). Lignins are difficult to break down by physical, chemical and other methods, and processing plant materials to release sugars from lignins requires extensive thermochemical treatment. In addition, lignin processing creates inhibitory byproducts, such as acetylated compounds, that hamper further extraction and fermentation. Acetyl esters released during treatment of cell wall polymers can inhibit saccharification of biomass. The released acetate is also inhibitory to the organisms used to ferment the sugars into useful byproducts. Overcoming plant recalcitrance to releasing biomaterials bound in the cell wall is therefore an issue of primary importance in the development of biofuel technology.
Lignins, complex interlinking biopolymers derived from hydroxyphenylpropanoids, provide rigidity and structure to plant cell walls for plant growth and transport of water and nutrients, and are significant contributors to plant recalcitrance. Lignins are composed primarily of syringyl (S), guaiacyl (G) and p-hydroxyphenyl (H) monolignol subunits, which are derived from sinapyl, coniferyl and p-coumaryl alcohols, respectively. The subunit ratio and resulting structure of plant lignins varies according to the genotype, environment, tissue type and maturity of the plant and as such, lignins are very heterogeneous and can vary significantly between different plants, within different tissues of a single plant and even within a single plant cell (Simmons B A et al., Curr Opin Plant Biol. 13:313-20 (2010)). This complexity and heterogeneity hinders the development of conversion technology able to process a range of sustainable feedstocks in a cost-effective manner.
Reduction of lignin biosynthesis, and decreases in cell wall recalcitrance, is desirable on one hand for biofuel production. Conversely, increases in cell wall recalcitrance and lignin biosynthesis can be desirable for production of lignin-based products such as carbon fibers. Thus, genetic manipulation of biomass feedstock to modulate lignin biosynthesis and sugar release hold promise both for production of improved, economically sustainable lignocellulosic biofuels (Vermerris W. et al., Crop Science 47(S3):S142-S153 (2007); Fu C. et al., PNAS 108:3803-3808 (2011)), for reducing processing costs for cellulose-based products such as pulp and paper or for enhancing the development of lignin-based polymers.
The genus Populus represents an economically important tree crop that has been targeted for use in diverse applications from the pulp and paper industry, carbon sequestration and as a feedstock in the lignocellulosic biofuel industry (Dinus R J. et al., Crit. Rev. Plant Sci. 20:51-69 (2001)). Recently, a study using wild Populus trichocarpa genotypes collected in the Pacific Northwest region demonstrated high phenotypic variation among the accessions in recalcitrance measured by lignin content and sugar release (Studer M H. et al., PNAS 108:6300-6305 (2011)). This study suggested that sufficient variation occurs in wild germplasm to identify specific genetic determinants of the recalcitrance trait by analysis of naturally-occurring allelic variability.
Quantitative trait loci (QTL) studies have been conducted using interspecific mapping of populations to identify genomic regions associated with cell wall phenotypes linked to recalcitrance (Novaes E. et al., New Phytologist 182:878-890 (2009); Yin T. et al., PLoS one 5:e14021 (2010)). Wegrzyn J L. et al., New Phytologist 188:515-532 (2010) and Muchero et al., BMC Genomics 16:4 (2015) demonstrated the feasibility of using linkage disequilibrium (LD)-based association mapping to validate candidate genes with putative functions in cell wall biosynthesis. The extent of LD decay in P. trichocarpa has been described by Slavov G T. et al., New Phytologist 196(3):713-25 (2012), who reported LD decay to below r2=0.2 within 2 kb in more than half of the genes, within a genomewide average 6-7 kb. Given that the average gene size for P. trichocarpa is 5 kb, these results suggest that QTL fine-mapping and association mapping to within single-gene resolution is possible in P. trichocarpa. 
Identification and manipulation of genes regulating cell wall biosynthesis and recalcitrance is critical both for efficient production of cellulosic sugars and ethanol from plant biomass, and for production of improved cellulose-based products, such as paper and pulp.